Process for preparing maytansinol

ABSTRACT

A novel Antibiotic C-15003 is produced by cultivating a microorganism of the genus Nocardia. 
     The Antibiotic C-15003 is useful as an antifungal agent or an antiprotozoan agent.

This is a division of application Ser. No. 131,787 filed Mar. 19, 1980,now U.S. Pat. No. 4,320,200, which in turn is a division of applicationSer. No. 972,492, filed Dec. 22, 1978, now U.S. Pat. No. 4,225,494,which is a division of application Ser. No. 811,448, filed June 29,1977, now U.S. Pat. No. 4,162,940.

This invention relates to Antibiotic C-15003, which is a novelantibiotic, a method of producing the same and a method of producingderivatives from said antibiotic.

We collected many soil and other samples and performed a screening ofthe microorganisms isolated from such samples. It was found, by thatscreening, that certain of the microorganisms were able to produce anovel antibiotic, that such microorganisms belonged to the genusNocardia and that by cultivating any of those microorganisms in asuitable medium, it was possible to have said antibiotic accumulated inthe cultured broth. It was also found that derivatives could be obtainedfrom said antibiotic. Further studies ensued, resulting in thedevelopment of this invention.

This invention is therefore directed to:

(1) Antibiotic C-15003 which has the general formula (I): ##STR1##

(2) A method of producing Antibiotic C-15003 characterized in that saidmethod comprises cultivating an Antibiotic C-15003-producing strain ofthe genus Nocardia in a medium to cause the strain to elaborate andaccumulate Antibiotic C-15003 in the cultured broth, and recovering saidAntibiotic C-15003 from said broth; and

(3) A method of producing a compound of the formula (II): ##STR2##characterized in that said method comprises hydrolyzing AntibioticC-15003 of the general formula (I): ##STR3##

In the context of this invention, the term "Antibiotic C-15003" means,generically, the three compounds having the above general formula (I) asa group, or a mixture of two or three of said compounds or, severally,any of the same compounds. Referring, also, to the general formula (I),the compound in which R is ##STR4## is referred to herein as "AntibioticC-15003 P-3" or more briefly as "C-15003 P-3"; the compound in which Ris --CO--CH₂ --CH₂ --CH₃ is referred to herein as "Antibiotic C-15003P-3'" or, more briefly, as "C-15003 P-3'"; the compound in which R is##STR5## is referred to herein as "Antibiotic C-15003 P-4" or, morebriefly, as "C-15003 P-4"; and the compound in which R is H[generalformula (II)] is referred to herein as "Antibiotic C-15003 P-0" or, morebriefly, as "C-15003 P-0".

As an example of the Antibiotic C-15003-producing strain ofmicroorganism, there may be mentioned an actinomycete Strain No. C-15003which we isolated from soil and other samples in our screening forantibiotic-producing microorganisms.

The microbiological characters of Strain No. C-15003 were investigatedby procedures analogous to those proposed by Schirling & Gottlieb[International Journal of Systematic Bacteriology 16, 313-340 (1966)].The results of observations at 28° C. over 21 days are as follows.

(1) Morphological characters

The vegetative mycelium extends well and develops into branches, both onagar and in liquid medium. Many of the hyphae measure 0.8 to 1.2 μm indiameter and, in certain instances, may divide into fragments resemblingrod bacteria or branched short lengths of hyphae. The strain gives goodgrowth on various taxonomical media, with aerial mycelium beingsuperimposed on the vegetative mycelium, although it frequently formscoremia like bodies (50-200×200-1000 μm) on which further aerial growthtakes place. Many of the aerial mycelia are flexuous, straight or aloosely spiral like configuration being encountered on a few occasions.Microscopic examination of aged cultures reveals that only in few casesthe conidia like cells occur in chains, while the cell suspensionsobtained from the surfaces of such cultures, as microscopicallyexamined, contained many elongated ellipsoidal (0.8-1.2 μm×4.8-6.8 μm)and ellipsoidal (0.8-1.2×1.0-2.0 μm) bodies resembling arthrospores.

Electron-microscopic examinations showed that these bodies had smoothsurfaces.

(2) The constituents of cells

The strain was shake-cultured in modified ISP No. 1 medium at 28° C. for66 to 90 hours, at the end of which time the cells were collected andrinsed. By the method of B. Becker et al. [Applied Microbiology 12, 421(1964)] and the method of M. P. Lechevalier. [Journal of Laboratory andClinical Medicine 71, 934 (1968)], the above whole cells were examinedfor diaminopimelic acid and sugar composition. The former was found tobe the meso-form, while spots were detected which corresponded togalactose and arabinose.

(3) Characteristics on taxonomical media

The strain showed comparatively good growth on various media, with thevegetative mycelium being colorless to pale yellow in initial phases ofculture and light yellowish tan to yellowish tan in later phases. Thestrain produces soluble pigments, yellow to yellowish tan, in varioustaxonomical media. The aerial mycelium is powdery and generally givesmoderate growth, being white to yellow or light yellowish tan. Thecharacteristics of the strain in various taxonomical media are set forthin Table 1.

                  TABLE 1                                                         ______________________________________                                        Cultural characteristics of Strain No. C-15003                                on taxonomical media                                                          ______________________________________                                        (A)    Sucrose nitrate agar:                                                         Growth (G): Moderate, Brite Melon Yellow (3 ia)*                              to Amber tan (3 lc)*, coremia like bodies                                     formed                                                                        Aerial mycelium (AM): Scant, white                                            Soluble pigment (SP): None or pale yellowish tan                       (B)    Glycerol nitrate agar:                                                        G: Moderate, Lt Ivory (2 ca)*, coremia like bodies                            formed                                                                        AM: Moderate, white                                                           SP: None                                                               (C)    Glucose asparagine agar:                                                      G: Moderate, Brite Marigold (3 pa)* to Brite Yellow                           (2 pa)*.                                                                      AM: Scant, white                                                              SP: Brite Yellow (2 pa)*                                               (D)    Glycerol asparagine agar:                                                     G: Moderate, Lt Ivory (2 ca)*, coremia like bodies                            formed                                                                        AM: Scant, white                                                              SP: None                                                               (E)    Starch agar:                                                                  G: Moderate, Lt Ivory (2 ca)* to Lt Wheat (2 ea)*,                            coremia like bodies formed                                                    AM: Abundant, Lt Ivory (2 ca)*                                                SP: None                                                               (F)    Nutrient agar:                                                                G: Moderate, Lt Ivory (2 ca)* to Colonial Yellow                              (2 ga)*, coremia like bodies formed                                           AM: Scant, white                                                              SP: None                                                               (G)    Calcium malate agar:                                                          G: Moderate Lt Ivory (2 ca)* to Lt Wheat (2 ea)*,                             coremia like bodies formed.                                                   AM: Moderate, white to Lt Ivory (2 ca)*                                       SP: None                                                               (H)    Yeast extract-malt extract agar:                                              G: Moderate, Amber (3 lc)* to Brite Yellow (3 la)*,                           coremia like bodies formed                                                    AM: Moderate, white to Lt Ivory (2 ca)*                                       SP: None                                                               (I)    Oatmeal agar:                                                                 G: Moderate, Lt Ivory (2 ca)* to Colonial Yellow                              (2 ga)*, coremia like bodies formed                                           AM: Scant, white to light yellow                                              SP: None                                                               (J)    Peptone yeast extract iron agar:                                              G: Moderate, Colonial Yellow (2 ga)*                                          AM: None                                                                      SP: Colonial Yellow (2 ga)*                                            (K)    Tyrosine agar                                                                 G: Moderate, Lt Ivory (2 ca)* to Lt Melon Yellow                              (3 ea)*, coremia like bodies formed.                                          AM: Moderate, white to Lt Ivory (2 ca)*.                                      SP: Camel (3 ie)*                                                      ______________________________________                                         *The color codes according to Color Harmony Manual, 4th ed. (Container        Corporation of America, 1958).                                           

(4) Physiological characters

The physiological characters of the strain are shown in Table 2.Temperature range for growth: 12° C. to 38° C. The temperature range inwhich good aerial growth occurs on agar (ISP No. 2) is 20° to 35° C.

                  TABLE 2                                                         ______________________________________                                        The physiological characters of Strain No. C-15003.                           ______________________________________                                        Temperature range for growth: 12 to 38° C.                             Temperature range for aerial growth: 20 to 35° C.                      Liquefaction of gelatin: Positive                                             Hydrolysis of starch: Positive                                                Reduction of nitrates: Positive                                               Peptonization of milk: Positive                                               Coagulation of milk: Negative                                                 Decomposition of casein: Positive                                             Production of melanoid pigments:                                                Negative (peptone yeast extract iron agar),                                   positive (tyrosine agar)                                                    Decomposition of tyrosine: Positive                                           Decomposition of xanthine: Negative                                           Decomposition of hypoxanthine: Negative                                       Tolerance to lysozyme: Positive                                               Tolerance to sodium chloride: 2%                                              ______________________________________                                    

(5) Utilization of various carbon sources

The utilization of various carbon sources was investigated using amedium described in Pridham and Gottlieb [Journal of Bacteriology 56,107 (1948)] and a basal medium of the same composition plus 0.1% ofyeast extract. The resultant spectrum is shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        The utilization of carbon sources by Strain No. C-15003                       Source of carbon                                                                          Growth    Sources of carbon                                                                           Growth                                    ______________________________________                                        D-Xylose    + ++*     Raffinose     ± ±*                                L-Arabinose + +       Melibiose     + +                                       D-Glucose   ++ ++     i-Inositol    - -                                       D-Galactose + +       D-Sorbitol    - -                                       D-Fructose  +++ ++    D-Mannitol    ++ ++                                     L-Rhamnose  + +       Glycerol      - ±                                    D-Mannose   +++ ++    Soluble starch                                                                              + +                                       Sucrose     ++ ++     Control       - -                                       Lactose     - -*                                                              Maltose     ± +                                                            Trehalose   + ++                                                              ______________________________________                                         *Basal medium with 0.1% yeast extract added                                   Note:                                                                         +++: Luxuriant growth                                                         ++: Good growth                                                               +: Growth                                                                     ±: Poor growth                                                             -: No growth                                                             

(6) Other characteristics

The cells were harvested by the procedure previously described in (2)and DNA was prepared by a procedure analogous to that of J. Marmur etal. [Journal of Molecular Biology 3, 208, 1961]. The G-C(Guanine-Cytosine) content of the DNA was found to be about 71 mole %.

Gram-staining of the vegetative mycelium of this strain was positive.

The above characteristics of Strain No. C-15003 were compared with thedescriptions in S. A. Waksman's "The Actinomycetes Vol.2"[The Williamsand Wilkins Co., 1961]; R. E. Buchanan and N. E. Gibbons, "Bergey'sManual of Determinative Bacteriology, 8th ed, 1974"; and otherliteratures.

Whilst this strain was thought to belong to Group III of the genusNocardia, the failure to find any species having the characters so fardescribed among the known strains led us to conclude that this strainrepresented a novel species of microorganism.

The present Strain No. C-15003 has been deposited at FermentationResearch Institute, Agency of Industrial Science and Technology (FERM)under the receipt number of 3992; at The Institute for Fermentation,Osaka (IFO) under the accession number of IFO 13726 and at The AmericanType Culture Collection (ATCC), Maryland, U.S.A. under the accessionnumber of 31281.

While Strain No. C-15003 is a novel species of the genus Nocardia asjust mentioned, it is liable, as are microorganisms generally, toundergo variations and mutations, either spontaneously or under theinfluence of a mutagen. For example, the many variants of the strainwhich are obtainable by irradiation with X-rays, gamma rays, ultravioletlight, etc., by monocell isolation, by culture on media containingvarious chemicals, or by any other mutagenic treatment, as well as themutants spontaneously derived from the strain, should not be consideredto represent any other distinct species but, rather, any of suchvariants and mutants capable of elaborating C-15003 P-3, P-3' and/or P-4may be invariably utilized for the purposes of this invention. By way ofexample, subjecting Strain No. C-15003 to various mutagenic treatmentsyields mutants substantially lacking the ability to produce solublepigments, mutants with substrate mycelia which are colorless, yellowishgreen, reddish tan or orange-red, mutants whose hyphae are ready tofragment into bacillary elements or branched short hyphal fragments, andmutants with abundant white aerial mycelia or substantially withoutaerial mycelia.

The medium employed for the cultivation of such an AntibioticC-15003-producing strain may be whichever of a liquid and a solid mediumonly if it contains nutrients which the strain may utilize, although aliquid medium is preferred for high-production runs. The medium maycomprise carbon and nitrogen sources which Strain No. C-15003 mayassimilate and digest, inorganic matter, trace nutrients, etc. Asexamples of said carbon sources may be mentioned glucose, lactose,sucrose, maltose, dextrin, starch, glycerol, mannitol, sorbitol, etc.,fats and oils (e.g. soybean oil, lard oil, chicken oil, etc.) and soforth. The nitrogen sources may for example be meat extract, yeastextract, dried yeast, soybean meal, corn steep liquor, peptone,cottonseed flour, spent molasses, urea, ammonium salts (e.g. ammoniumsulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.)and so forth. The medium may further contain salts of sodium, potassium,calcium, magnesium, etc. salts of iron, manganese, zinc, cobalt, nickel,etc., salts of phosphoric acid, boric acid, etc., and organic acid saltssuch as acetates and propionates. Further, the medium may contain, asadded, various amino acids (e.g. glutamic acid, aspartic acid, alanine,glycine, lysine, methionine, proline, etc.), peptides (e.g. dipeptides,tripeptides, etc.), vitamins (e.g. B₁, B₂, nicotinic acid, B₁₂, C, E,etc.), nucleic acids, (e.g. purine, pyrimidine and derivatives thereof)and so forth. For the purpose of adjusting the pH of the medium, theremay be added an inorganic or organic acid, alkali, buffer or the like.Suitable amounts of oils, fats, surfactants, etc. may also be added asantifoams.

The cultivation may be conducted by any of the stationary, shake,submerged aerobic and other cultural conditions. For high productionruns, submerged aerobic culture is of course preferred. While theconditions of culture, of course, depends upon the condition andcomposition of medium, the strain, cultural method and other factors, itis normally preferred to carry out incubation at 20° to 35° C. with aninitial pH of about 7.0 or thereabouts. Particularly desirable is atemperature from 23° to 30° C. in an intermediate stage of cultivation,with an initial pH of 6.5 to 7.5. While the incubation time also isvariable according to the same factors as mentioned above, it isadvisable to continue the incubation until the titer of the desiredantibiotic product becomes maximal. In the case of shake culture oraerobic submerged culture in liquid medium, the time required normallyranges from about 48 to 144 hours.

The potency of the antibiotic was assayed with Tetrahymena pyriformis Was an assay organism. Thus, the above microorganism was grown on a testmedium [20 g of Proteose-peptone (Difco), 1 g of yeast extract (Difco),2 g of glucose, 1000 ml of distilled water and 10 ml of 1 M-phosphatebuffer (pH 7.0)] at 28° C. for 44 to 48 hours and the potency of theantibiotic was determined by the serial dilution method wit a moniteringof the turbidity of growth, effect on ascites tumor cells and by athin-layer chromatographic (briefly TLC) assay to be describedhereinafter.

The novel Antibiotic C-15003 P-3, P-3' and/or P-4 is produced andaccumulated in the resultant fermentation broth, both extracellularlyand intracellularly,

These substances have also been detected by TLC. Thus, the fermentationbroth is separated into cells and filtrate by filtration oncentrifugation and the filtrate is extracted with the same volume ofethyl acetate. To the cells is added the same amount of 70%acetone-water as the filtrate and, after an hour's stirring at 20° C.,the suspension is filtered. The acetone is removed from the filtrate andthe resultant aqueous filtrate is extracted with ethyl acetate. Each ofthe extracts is concentrated to 1/100 by volume and subjected tothin-layer chromatography on a silica gel-glass plate (Merck, WestGermany, Kieselgel 60 F 254, 0.25 mm, 20×20) (solvent system:chloroform-methanol=9:1). The potency was determined on the basis of theintensity of spots detected by irradiation with ultraviolet light at2537 A.

Because C-15003 P-3, P-3' and/or P-4, which are thus produced in thefermentation broth, are lipophyl neutral substances, they can beconveniently recovered by separation and purification procedures whichare normally employed for the harvest of such microbial metabolites. Forexample, there may be employed a procedure which utilizes the differencein solubility between the antibiotic and impurity, means which utilizesthe adsorptive affinity of various adsorbents such as activated carbon,macroporous nonionic resins, silica gel, alumina, etc., a procedure ofremoving the impurities by means of ion exchange resins, and so forth,as applied singly or in a suitable combination or as applied inrepetition.

Since, as aforesaid, C-15003 P-3, P-3' and P-4 occur in both thefiltrate and cells, the antibiotics are separated and purified by meansof such an adsorbent, if one is employed, either directly or after asolvent extraction in the case of the filtrate, or after a solventextraction in the case of microbial cells. The solvent extraction may beperformed by any of the following and other methods e.g. (1) solventextraction from the culture broth prior to separation of cells and (2)solvent extraction of the cells and the filtrate obtained by filtration,centrifugation or other process. To extract the filtrate and cellsindependently, the following procedure may be taken advantageously.

The solvents suitable for extraction of the filtrate arewater-immiscible organic solvents such as fatty acid esters, e.g. ethylacetate and amyl acetate; alcohols, e.g. butanol; halogenatedhydrocarbons, e.g. chloroform; and ketones, e.g. methyl isobutyl ketone.The extraction is carried out at a pH near neutral and, preferably, theculture fluid previously adjusted to pH 7 is extracted with ethylacetate. The extract is washed with water and concentrated under reducedpressure. Then, a nonpolar solvent such as petroleum ether or hexane isadded to the concentrate and the crude product I containing the activecompound is recovered. Because, on TLC, a number of spots are detectedin addition to Antibiotic C-15003, the product I is sequentiallysubjected to the following purification procedures. Thus, as a routinepurification procedure, adsorption chromatography is useful and, forthis purpose, one of those common adsorbents such as silica gel,alumina, macroporous nonionic adsorbent resin, etc. may be employed. Forpurification from the crude product I, silica gel is most useful. Anddevelopment may be carried out, for example starting with petroluemether and hexane and elution of Antibiotic C-15003 is performed by theaddition of a polar solvent such as ethyl acetate, acetone, ethanol ormethanol. In a typical process, using silica gel (Merck, West Germany,0.05-0.2 mm) as a carrier, column chromatography is carried out with aserial increase in the hexane to ethyl acetate ratio. The eluate issampled and investigated by TLC and the fractions containing C-15003 arepooled and concentrated under reduced pressure. Then, petroleum ether orhexane is added to the concentrate, whereby the crude product II isobtained. Since this product still contains impurities, it is furtherpurified as follows. For example, the product II may be purified bymeans of a second silica gel column using a different solvent system.The developing system for this purpose may consist in a halogenatedhydrocarbon such as dichloromethane or chloroform, with the addition ofa polar solvent such as an alcohol, e.g. methanol or ethanol, a ketone,e.g. acetone or methyl ethyl ketone, or the like. In this way,Antibiotic C-15003 is isolated. The order of solvent systems for thefirst and second silica gel columns may be reversed and, in addition,ordinary organic solvents may be used in conjunction with the abovesystems if necessary.

Where a macroporous adsorbent resin is used as purification means forcrude product II, elution of Antibiotic C-15003 is accomplished with amixture of water with a lower alcohol, a lower ketone or an ester. Thelower alcohol may for example be methanol, ethanol, propanol or butanoland the lower ketone may for example be acetone or methyl ethyl ketone.The ester may for example be ethyl acetate. In a typical procedure, thecrude product II is dissolved in 60% methanol-water and adsorbed on acolumn of Diaion HP-10 (Mitsubishi Kasei K.K.). The column is washedwith 70% methanol-water and, then, elution is carried out with 90%methanol-water. In this way, Antibiotic C-15003 is eluted from thecolumn.

In either of the processes described above, the fractions containingAntibiotic C-15003 are pooled and concentrated under reduced pressure.To the dry product is added 5 to 8 volumes of ethyl acetate and themixture is allowed to stand, whereupon crystals of Antibiotic C-15003separate. These crystals contain C-15003 P-3, P-3' and P-4. Thesecompounds are then separated from each other by means of an adsorbentsuch as those mentioned hereinbefore. Thus, using silica gel or amacroporous nonionic adsorbent resin and the above solvents, the desiredcompounds may be fractionally eluted. When, for example, silica gel isemployed, development is carried out with hexane, ethyl acetate, orchloroform-methanol, whereby C-15003 P-4, P-3' and P-3 emerge in thatorder. After detection by TLC, the fractions corresponding to C-15003P-4, P-3' and P-3 are respectively concentrated under reduced pressureand ethyl acetate is added to the concentrates. In this manner, therespective compounds can be obtained as crystals. When a macroporousnonionic adsorbent resin is employed, gradient elution with a varyingratio of alcohol, ketone or ester to water may be utilized. For example,by the gradient elution method involving the use of 60% methanol-waterand 95% methanol-water, with 5% sodium chloride added, C-15003 P-3, P-3'and P-4 merge in the order mentioned. After sampling and detection byTLC, each group of active fractions is concentrated under reducedpressure and crystallized from ethyl acetate. The isolated crystalsinclude ethyl acetate as a solvent of crystallization and, after dryingover phosphorus pentoxide at 70° C. for 8 hours, show the followingphysical and chemical properties. (Table 4).

                                      TABLE 4                                     __________________________________________________________________________              Antibiotic C-15003                                                            P - 3        P - 3'       P - 4                                               C.sub.32 H.sub.43 ClN.sub.2 O.sub.9 = 635.169                                              C.sub.32 H.sub.43 ClN.sub.2 O.sub.9                                                        C.sub.33 H.sub.45 ClN.sub.2 O.sub.9 =                                         649.196                                   __________________________________________________________________________    Melting point (°C.)                                                              190-192°                                                                            182-185°                                                                            177-180°                           Specific rotation                                                                       -136° ± 10°                                                               -134° ± 10°                                                               -142° ± 10°              (α).sub.D.sup.22°                                                          (C = 0.375 CHCl.sub.3)                                                                     (C = 0.11 CHCl.sub.3)                                                                      (C = 0.522 CHCl.sub.3)                    Elemental analysis                                                                      C     60.06  60.09        60.65                                     Found (%) H     7.04   7.02         7.05                                                N     4.33   4.34         4.25                                                Cl    5.37   5.99         5.23                                      Elemental analysis                                                                      C     60.51  60.51        61.05                                     Calcd. (%)                                                                              H     6.82   6.82         6.99                                                N     4.41   4.41         4.32                                                Cl    5.58   5.58         5.46                                      Ultraviolet                                                                             233(30250) 240(sh 28450)                                                                   233(30155) 240(sh 28250)                                                                   233(29900) 240(sh 28240)                  absorption spectra                                                                      252(27640) 280(5750)                                                                       252(27600) 280(5750)                                                                       252(27590) 280(5712)                      nm(ε)                                                                           288(5700)    288(5700)    288(5680)                                 (in methanol)                                                                 Infrared  1740, 1730, 1670, 1580                                                                     1740, 1730, 1670, 1580,                                                                    1740, 1730, 1670, 1580                    absorption spectra                                                                      1445, 1385, 1340, 1255                                                                     1445, 1385, 1340, 1255,                                                                    1445, 1385, 1340, 1255                    (cm.sup.-1)KBr                                                                          1180, 1150, 1100, 1080,                                                                    1180, 1150, 1100, 1080,                                                                    1180, 1150, 1100, 1080,                             1038         1038         1038                                      Nuclear magnetic                                                                        1.27(d) (3H) 1.06(t) (3H) 1.03(d) (6H)                              resonance spectra                                                                       1.28(d) (3H)                                                        (ppm)                                                                         100MHz in CDCl.sub.3                                                          Mass spectra(m/e)                                                                       573, 485, 470, 450                                                                         573, 485, 470, 450                                                                         587, 485, 470, 450                        Solubility                                                                              Insoluble in petr. ether,                                                                  Insoluble in petr. ether,                                                                  Insoluble in petr. ether,                           hexane & water.                                                                            hexane & water.                                                                            hexane & water.                                     Sparingly soluble in                                                                       Sparingly soluble in                                                                       Sparingly soluble in                                benzene & ether.                                                                           benzene & ether.                                                                           benzene & ether.                                    Soluble in chloroform,                                                                     Soluble in chloroform,                                                                     Soluble in chloroform,                              ethyl acetate, acetone,                                                                    ethyl acetate, acetone,                                                                    ethyl acetate, acetone,                             ethanol, methanol,                                                                         ethanol, methanol,                                                                         ethanol, methanol,                                  pyridine, tetrahydrofuran                                                                  pyridine, tetrahydrofuran                                                                  pyridine, tetrahydrofuran                           & dimethylsulfoxide                                                                        & dimethylsulfoxide.                                                                       & dimethylsulfoxide.                      Color reactions                                                                         Dragendorff: Positive                                                                      Dragendorff: Positive                                                                      Dragendorff: Positive                               Beilstein: Positive                                                                        Beilstein: Positive                                                                        Beilstein: Positive                       __________________________________________________________________________

Based on the above molecular formula shown above and the antimicrobialand antitumor activity data given hereinafter, the present antibioticwas compared with the known groups of antibiotics. The literature searchfailed to locate no distinct group similar to Antibiotic C-15003.However, a search for substances that might give ultraviolet absorptionssimilar to those of the present antibiotic among component of plant andother naturally-occurring organic compounds led us to the maytanacinegroups and, based on the molecular formulas involved, in particular, itwas assumed that the antibiotic belongs to the maytanacine group ofcompounds containing two nitrogen atoms. Maytanacine was obtained as acomponent of plant and was reported in Journal of American ChemicalSociety 97, 5294(1975). The mass spectrum of maytanacine is as follows.

    ______________________________________                                        M.sup.+ -(a)                                                                          M.sup.+ -(a + b)                                                                             485-CH.sub.3                                                                           485-Cl                                        545     485            470      450                                           ______________________________________                                         (a) = H.sub.2 O + HNCO                                                        ##STR6##                                                                     -  The presence of m/e 485, 470 and 450 for C-15003 P-3, P-3' and P-4          convinces us at once that these compounds have a skeletal structure     identical with that of maytanacine, differentiating them from maytanacine     in the kind of acyl group in 3-position. It is thus clear that Antibiotic     C-15003 is a novel compound. When C-15003 P-3, P-3' and P-4 were each     degradated with alkali and analyzed by gas chromatography for the     liberated carboxylic acids, it was found that isobutyric acid, butyric     acid and isovaleric acid were obtainable from C-15003 P-3, C-15003 P-3'     and C-15003 P-4, respectively. FIG. 1 shows the structures, based on the     above data, of C-15003 P-3, P-3' and P-4.

    __________________________________________________________________________    FIG. 1                        R                                               __________________________________________________________________________     ##STR7##                                                                                                  ##STR8##                                         __________________________________________________________________________

Biological activity

(A) Antimicrobial activity:

With trypticase-soy agar (BBL) as an assay medium, the inhibitoryconcentrations against the microorganisms named below were investigatedby the paper disc method. Thus, filter-paper discs (Toyo Seisakusho,thin-type, 8 mm dia.) each impregnated with 0.02 ml of a 300 μg/mlsolution of C-15003 P-3, P-3' or P-4 were placed on plates respectivelyinoculated with the microorganisms named below to investigate theminimal inhibitory concentrations. The results showed that theantibiotics had no activity against the following microorganisms:Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonasaeruginosa, Staphylococcus aureus, Bacillus subtilis, Bacillus cereus,Klebsiella pneumoniae, Serratia marcescens, Mycobacterium avium.

On the other hand, with agar plates containing the assay medium [3.5 gdisodium hydrogen phosphate, 0.5 g monopotassium dihydrogen phosphate, 5g yeast extract (Difco), 10 g glucose, 15 g agar, 1000 ml distilledwater, pH 7.0], the growth inhibition against Talaromyces avellaneus wasassayed. In this assay, the minimal inhibitory concentrations were 3μg/ml for C-15003 P-3 and P-3', and 1.5 μg/ml for C-15003 P-4.Furthermore, the wild strain of Tetrahymena pyriformis W as an assayorganism was cultivated on an assay medium [composed of 20 gProteose-peptone (Difco), 1 g yeast extract, 2 g glucose, 1000 mldistilled water and 10 ml 1 M-phosphate buffer pH 7.0] at 28° C. for 44to 48 hours and the growth inhibitory activity of the antibioticcompounds against this particular microorganism was determined by theserial dilution method. Growth inhibition occurred at 1 μg/ml forC-15003 P-3 and P-3', and at 0.5 μg/ml for C-15003 P-4.

Anti-fungal activity is shown in Table 5. As seen from Table 5, C-15003has growth inhibitory activity against microorganisms which cause plantdiseases. The filter-paper discs impregnated with 0.02 ml of a 1000μg/ml solution of C-15003 were placed on plates respectively inoculatedwith the microorganisms as following Table 5.

                  TABLE 5                                                         ______________________________________                                        Anti-microbial spectra                                                                       IFO                    Inhibition                              Test organisms number   medium   hour diameter                                ______________________________________                                        Alternaria     7515     PSA*     48   38                                      kikuchiana                                                                    Fusicladium levieri                                                                          6477     PSA*     90   68                                      Helminthosporium                                                              sigmoideum var.                                                                              5273     PSA*     48   55                                      irregulare                                                                    Pyricularia oryzae                                                                           --       PSA*     48   53                                      Elsinoe fawcetti                                                                             8417     PSA*     90   55                                      Fusarium oxysporum                                                                           --       PSA*     48   20                                      f. cucumerinum                                                                Guignardia laricina                                                                          7888     PSA*     48   12                                      Cochlioborus miyabeanus                                                                      5277     PSA*     48   60                                      Diaporthe citri                                                                              9170     PSA*     48   55                                      Gibberella zeae                                                                              8850     PSA*     48   37                                      Sclerotinia sclerotiorum                                                                     9395     PSA*     90   65                                      Venturia pirina                                                                              6189     PSA*     48   50                                      Pellicularia sasakii                                                                         9253     PSA*     48   50                                      Pythium aphanidermatum                                                                       7030     PSA*     48   58                                      Botrytis cinerea                                                                             --       PSA*     48   48                                      Aspergillus niger                                                                            4066     PSA*     48   0                                       Penicillium chrysogenum                                                                      4626     PSA*     48   35                                      Rhizopus nigricans                                                                           6188     PSA*     48   25                                      Saccharomyces cerevisiae                                                                     0209     PSA*     48   0                                       Rhodotorula rubra                                                                            0907     PSA*     48   28                                      Trichophyton rubrum                                                                          5467     GB**     48   38                                      Trichophyton                                                                   mantagrophytes                                                                              7522     GB**     48   38                                      Candida albicans                                                                             0583     GB**     48   0                                       Candida utilis 0619     GB**     48   0                                       Cryptococcus   0410     GB**     48   43                                      neoformans                                                                    ______________________________________                                         *PSA: Potato sucrose agar medium                                              **GB: Glucose nutrient agar medium                                       

(B) Antitumour activity

The therapeutic effects of C-15003 P-3, P-3' and P-4 (dosedintraperitoneally for 9 consecutive days) upon P388 leukemia in mice(1×10⁶ cells/animal, mouse, intraperitoneally transplanted) wereinvestigated. The results showed that, in terms of life span-extendingratio, these compounds had an antitumour activity as high as 200% at thedose level of 0.00625 mg/kg/day.

(C) Toxicity

In an acute toxicity test with mice as test animals, which involvedintraperitoneal injections of C-15003 P-3, P-3' and P-4, all of theseantibiotics showed a LD₅₀ value more than 0.313 mg/kg.

As mentioned hereinbefore, the present Antibiotic C-15003 has stronginhibitory activity against fungi and protozoa and, therefore, is ofvalue as an antifungal or antiprotozoan agent. Furthermore, becauseAntibiotic C-15003 displays a life span-extending action upontumour-bearing mammalian animals (e.g. mouse), it is also expected thatthe compound will be of use as an antitumour drug.

Antibiotic C-15003, as an antifungal and antiprotozoan agent, can beused with advantage for an assessment of the bacterial ecology in thesoil, active sludge, animal body fluid or the like. Thus, when valuablebacteria are to be isolated from soil samples or when the actions ofbacteria are to be evaluated independently of those of fungi andprotozoa in connection with the operation and analysis of an activesludge system used in the treatment of waste water, as the presentantibiotic may be utilized to obtain a selective growth of the bacterialflora without permitting growth of the concomitant fungi and protozoa inthe specimen. In a typical instance, the sample is added to a liquid orsolid medium and 0.1 ml of a 10 to 100 μg/ml solution of the antibioticin 1% methanol-water is added per ml of the medium, which is thenincubated.

The present Antibiotic C-15003 can also be used as an anti-microbialagent for the treatment of plant diseases caused by the microorganismsmentioned in the above Table 5.

In the typical application, Antibiotic C-15003 is used in a form of 1%methanolic aqueous solution containing 0.5 μg/ml--5 μg/ml of theantibiotic. For instance Antibiotic C-15003 may be used for the controlof the reddish brown sheath rot, the blast, the Helminthosporium leafspot and the sheat blight of rice plants.

It is thus apparent that C-15003 P-3, P-3' and P-4 are all novelcompounds having the same skeletal structure and can be used also asintermediates for the production of other pharmaceutically usefulcompounds. Thus, by way of deacylation reaction, C-15003 P-O[maytansinol] with a hydroxyl group in 3-position can be derived fromthe present antibiotic. In this connection, because the acyl group is inposition beta to carbonyl, the conventional reductive hydrolysisreaction can be employed with advantage. Thus, by means of a complexmetal hydride [e.g. lithium aluminum hydride (LiAlH₄)] at a lowtemperature (e.g. -20°-0° C.), the O-ester bond in 3-position may behydrolytically cleaved, without affecting other functional groups, e.g.carbonyl, epoxy, carbon-carbon double bonds, etc., to yield maytansinol.The physical and chemical data on this maytansinol sample thus obtainedis in good agreement with the data given in Kupchan et al., The Journalof American Chemical Society 97, 5294-5295 (1975)].

The following examples are further illustrative but by no meanslimitative of the invention, wherein "part(s)" is based on weight unlessotherwise noted and the relationship between "part(s)" and "part(s) byvolume" corresponds to that between "gram(s)" and "milliliter(s)", and"%" is based on "weight/volume" unless otherwise noted.

EXAMPLE 1

Nocardia No. C-15003 (IFO 13726; FERM 3992; ATCC 31281) as grown on amedium (yeast extract-melt extract agar) was used to inoculate a 200parts by volume fermenter containing 40 parts by volume of a seedculture medium (2% glucose, 3% soluble starch, 1% raw soybean meal, 1%corn steep liquor, 0.5% Polypepton, 0.3% NaCl, 0.5% CaCO₃, pH 7.0). Theinoculated medium was incubated at 28° C. for 48 hours to obtain aninoculum. A 0.5 part by volume portion of the inoculum thus obtained wastransferred to a 200 parts by volume fermenter containing 40 parts byvolume of a fermentation medium composed of 5% dextrin, 3% corn steepliquor, 0.1% Polypepton and 0.5 CaCO₃ (pH 7.0), and cultivated at 28° C.for 90 hours to give inoculum (seed culture).

As determined by the serial dilution method using Tetrahymena pyriformisW as an assay organism and Antibiotic C-15003 P-3 as the standardsample, the above culture was found to have a titer of 25 μg/ml.

EXAMPLE 2

A 10 parts by volume portion of the inoculum (seed) obtained in Example1 was transferred to a 2000 parts by volume fermenter containing 500parts by volume of a seed culture medium (same as above) and incubatedat 28° C. for 48 hours. A 500 parts by volume portion of the resultantculture was transferred to a 50,000 parts by volume tank of stainlesssteel containing 30,000 parts by volume of seed culture medium andcultivated at 28° C., under aeration (30,000 parts by volume/min.),agitation [280 r.p.m. (178 DT)] and internal pressure (1 kg/cm²) toobtain a seed culture. This culture was used to seed a 200,000 parts byvolume tank of stainless steel containing 100,000 parts by volume of afermentation medium similar to the one used in Example 1 at aninoculation rate of 10%. The inoculated medium was incubated at 28° C.,under aeration (100,000 parts by volume/min.), agitation [200 r.p.m.(1/2DT) and internal pressure (1 kg/cm²) for 90 hours. As determined bythe same procedure as that described in Example 1, the culture obtainedabove was found to have a titer of 25 μg/ml.

EXAMPLE 3

To 95,000 parts by volume of the culture obtained in Example 2 was added2,000 parts of Hyflo-supercel® (Johnes and Manville Products, U.S.A.)and, after thorough mixing, the mixture was filtered on a pressurefilter to obtain 85,000 parts by volume of filtrate and 32,000 parts ofmoist cells. The filtrate 85,000 parts by volume was stirred andextracted with 30,000 parts by volume of ethyl acetate. This procedurewas repeated once again. The ethyl acetate layers were pooled, washedtwice with 30,000 parts by volume portions of water, dried by theaddition of 500 parts of anhydrous sodium sulfate and concentrated underreduced pressure to 200 parts by volume. Petroleum ether was added tothe concentrate and the resultant precipitate was recovered byfiltration (53 parts). This crude product I was stirred with 100 partsby volume of ethyl acetate and the insolubles were filtered off. Thefiltrate was stirred with 10 parts of silica gel (Merck, West Germany,0.05-0.2 mm) and the ethyl acetate was removed under reduced pressure.The residue was applied to the top of a silica gel column (400 parts byvolume). Elution was carried out with 500 parts by volume of hexane, 500parts by volume of hexane-ethyl acetate (3:1), 500 parts by volume ofhexane-ethyl acetate (1:1), 500 parts by volume of hexane-ethyl acetate(1:3), 500 parts by volume of ethyl acetate and 1,000 parts by volume ofethyl acetate-methanol (50:1), with the eluate being collected in 100parts by volume fractions.

One part by volume portion of each fraction was concentrated to dryness,and 0.1 part by volume of ethyl acetate was added to the concentrate togive a mixture. The mixture was spotted at 2.5 cm from the bottom edgeof a silica gel-glass plate (Merck, West Germany, 60 F ₂₅₄, 0.25 mm,20×20) and developed for about 17 cm with a solvent system of ethylacetate-methanol (19:1). After development, detection was carried outwith ultraviolet light (2537A).

The active fractions No. 23-No.28 of Rf 0.6-0.65 were collected andconcentrated under reduced pressure to about 20 parts by volume. To thisconcentrate was added 150 parts by volume of petroleum ether to obtain15 parts of a crude product II.

EXAMPLE 4

With stirring, 32,000 parts of the moist cells obtained in Example 3were extracted with 50,000 parts by volume of 70% acetone-water for 3hours and, then, filtered on a pressure filter. The extraction with50,000 parts by volume of 70% acetone-water and subsequent filtrationwas repeated once again. The filtrates were pooled and the acetone wasremoved by concentration under reduced pressure. The resultant aqueoussystem was passed through a column of 5,000 parts by volume Diaion HP-10(Mitsubishi Kasei K.K.). The column was washed with 20,000 parts byvolume of water and 50% aqueous methanol, followed by elution with15,000 parts by volume of 90% methanol-water. The eluate wasconcentrated under reduced pressure to 3,000 parts by volume and shakenwith 3,000 parts by volume of water and 3,000 parts by volume of ethylacetate. The above procedure was repeated once again. The ethyl acetatelayers were combined, washed with water, dried by the addition ofanhydrous sodium sulfate and concentrated under reduced pressure to 200parts by volume. Following the addition of petroleum ether, theprecipitate was recovered by filtration (28 parts). The above productwas purified by means of a column of silica gel to recover 8.0 parts ofcrude product II.

EXAMPLE 5

In 10 parts by volume of ethyl acetate was dissolved 1.5 parts of thecrude product II obtained in Example 3 and the solution was stirred wellwith 4 parts of silica gel (Merck, West Germany, 0.05-0.2 mm). The ethylacetate was removed under reduced pressure. The residue was applied tothe top of a column of 300 parts by volume silica gel and the column wasfirst washed with 500 parts by volume of chloroform and then eluted with500 parts by volume of chloroform-methanol (50:1), 500 parts by volumeof chloroform-methanol (20:1) and 500 parts by volume ofchloroform-methanol (10:1). The eluate was collected in 25 parts byvolume fractions.

A 0.5 part by volume portion of each fraction was concentrated underreduced pressure. To the concentrate was added 0.05 part by volume ofethyl acetate, and the mixture as a sample was subjected to silica gelthin layer chromatography (developing system: chloroform-methanol=9:1).

The fractions Nos. 39 and 40 absorbing at 2537 A in the zone of Rf0.50-0.60 were collected and concentrated to dryness under reducedpressure. To the residue was added 2 parts by volume of ethyl acetateand the mixture was allowed to stand, whereupon 0.150 part crystals ofAntibiotic C-15003 were obtained.

The above crystals of Antibiotic C-15003 (0.150 part) were dissolved in15 parts by volume of methanol, followed by addition of 0.300 part ofsodium chloride and 15 parts by volume of water. A column was packedwith 200 parts by volume of Diaion HP-10 (Mitsubishi Kasei K.K.) andcalibrated with 600 parts by volume of 50% methanol-water containing 5%of NaCl. The sample solution prepared above was passed through thecolumn, and gradient elution was carried out using 1,500 parts by volumeof 60% methanol-water containing 5% NaCl and 1,500 parts by volume of95% methanol-water. The eluate was collected in 15 parts by volumefractions and each fraction was investigated by silica gel thin layerchromatography. The fractions 145 to 153 contained C-15003 P-3, thefractions 167-180 contained C-15003 P-3' and P-4 and the fractions185-190 contained C-15003 P-4.

Each group of fractions was concentrated and dissolved by the additionof 50 parts by volume of water and 100 parts by volume of ethyl acetate.The solution was shaken in a separatory funnel and the water layer wasseparated and, after washing with two 50 parts by volume-portions ofwater, the ethyl acetate layer was dried over anhydrous sodium sulfate,concentrated and allowed to stand. In the above manner, crystals wereobtained from each group of fractions. The crystals were collected byfiltration and dried.

    ______________________________________                                        C-15003 P-3           0.070 part                                              C-15003 P-3', P-4     0.018 part                                              C-15003 P-4           0.015 part                                              ______________________________________                                    

The mixed crystals of C-15003 P-3' and P-4 (0.018 part) were dissolvedin 0.3 part by volume of ethyl acetate and spotted in a line at adistance of 2.5 cm from the bottom edge of a silica gel glass plate(Merck, West Germany, Kieselgel 60 F₂₅₄ 0.25 mm, 20×20), followed bydevelopment with ethyl acetate-methanol (19:1). After development toabout 18 cm, the absorption band at Rf 0.68 (P-4) and Rf 0.65 (P-3')were scrapped off and each was independently extracted twice with ethylacetate containing a small amount of water. The resultant ethyl acetateextract was washed with water, dried over anhydrous sodium sulfate,concentrated under reduced pressure and allowed to stand.

0.010 Part crystals of C-15003 P-4 and 0.003 part crystals of C-15003P-3' were obtained from the fractions of Rf 0.68 and Rf 0.65,respectively.

EXAMPLE 6

One thousand parts by volume of the culture of Example 2 was inoculatedinto a 200,000 parts by volume tank of stainless steel containing100,000 parts by volume of a seed culture medium and the inoculatedmedium was incubated at 28° C. under aeration (100,000 parts byvolume/min.) and agitation (200 r.p.m.) for 48 hours to prepare a seedculture. This seed culture was transferred to a 2,000,000 parts byvolume tank of stainless steel containing 1,000,000 parts by volume of afermentation medium similar to that used in Example 1 at atransplantation rate of 10%. Cultivation was carried out at 28° C. underaeration (1,000,000 parts by volume/min.), agitation [120 r.p.m. (1/3DT)] and internal pressure (1 kg/cm²) for 90 hours. The resultantculture was found to have a titer of 20 μg/ml as assayed by the assayprocedure described in Example 1.

To 900,000 parts by volume of the above culture was added 900,000 partsby volume of acetone and, after an hour's stirring, 20,000 parts ofHyflo-Supercel (Johnes & Manville, U.S.A.) was added. The mixture wasfurther stirred and filtered on a pressure filter machine.

To 1,700,000 parts by volume of the resultant filtrate was added 500,000parts by volume of water and, in a Podbielniak (Podbielniak, Inc.), themixture was extracted with 1,000,000 parts by volume of ethyl acetate.The ethyl acetate layer was washed with water, dried by the addition ofanhydrous sodium sulfate and concentrated under reduced pressure. To theconcentrate was added petroleum ether and the resultant precipitate wasrecovered by filtration and dried. By the above procedure was obtained68 parts of crude product I. Thereafter, as in Examples 3, 4 and 5, thiscrude product was purified to obtain 9.5 parts of C-15003 P-3, 0.300part of C-15003 P-3' and 2.5 parts of C-15003 P-4.

EXAMPLE 7

In 1 part by volume of tetrahydrofuran was dissolved 0.015 part of theAntibiotic C-15003 crystals obtained in Example 5 and after the solutionwas cooled to -5° C., 0.012 part of lithium aluminum hydride was added.The mixture was allowed to stand for 2 hours. Following the addition of0.5 part by volume of a 1% aqueous solution of H₂ SO₄, the reactionmixture was extracted with 2 parts by volume of ethyl acetate. The ethylacetate layer was washed with water, dried by the addition of anhydroussodium sulfate and concentrated under reduced pressure. Preparative TLCwith silica gel was carried out on the concentrate and the zone of Rf0.25 to 0.3 was scraped off and extracted with ethyl acetate containinga small amount of water. The extract was washed with water, dried overanhydrous sodium sulfate and concentrated under reduced pressure,whereupon crystals separated. The crystals were recovered by filtrationand dried. By the above procedure was obtained 0.010 part of C-15003P-0, melting point 174° C.

Elemental analysis: Found C, 59.65; H, 6.58; N, 5.02; Cl, 6.51; calcd.for C₂₈ H₃₇ ClN₂ O₈ C, 59.52; H, 6.60, N, 4.96; Cl, 6.27.

IR: 1715, 1670, 1580(cm⁻¹)

UV(nm): 232(32,750), 244(sh, 30,850), 252(31,650), 281(5750), 288(5700)

In properties, this product is identical with maytansinol.

What we claim is:
 1. A method of producing a compound of the formula:##STR9## which comprises subjecting Antibiotic C-15003 of the formula:##STR10## wherein R represents ##STR11## --CO--CH₂ --CH₂ --CH₃ or##STR12## to reductive hydrolysis.
 2. A method as claimed in claim 1,wherein the reductive hydrolysis is conducted by employing a complexmetal hydride.
 3. A method as claimed in claim 2, wherein the complexmetal hydride is lithium aluminum hydride.